Methods and Compositions for the Diagnosis and Treatment of Cancer

ABSTRACT

The present invention relates to methods, kits, and compositions for detecting and/or diagnosing metastatic potential of cancer cells or for evaluating prognosis in a patient with cancer by detection of the protein expression level of an HLA class I molecule and/or the copy number variation of a polynucleotide encoding the HLA class I molecule. The present invention also relates to the use of the protein expression level of an HLA class I molecule and/or the copy number variation of a polynucleotide encoding the HLA class I molecule as a prognosis biomarker and metastasis predictive biomarker of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims benefit under 35 USC 119(e) of ChineseApplication No. 201410512479.7 filed Sep. 29, 2014, which isincorporated by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named469601_SEQLST.txt, created on Dec. 10, 2015, and containing 1,743 bytes,which is hereby incorporated by reference in its entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates to methods, kits, and compositions fordetecting and/or predicting metastatic potential of gastric cancer cellsor for evaluating prognosis in a patient with gastric cancer bydetection of the protein expression level of an HLA class I moleculeand/or the copy number variation of a polynucleotide encoding the HLAclass I molecule combined with the number of NK cells at the tumor site.The present invention also relates to the use of the protein expressionlevel of an HLA class I molecule and/or the copy number variation of apolynucleotide encoding the HLA class I molecule as a prognosisbiomarker and metastasis predictive biomarker of cancer.

BACKGROUND OF THE INVENTION

Gastric cancer is among the malignancies with highest morbidityworldwide. Most patients with gastric cancer die of metastasis orrelapse, leading to high mortality of the disease. Compared with otherorgans, the stomach tissue contains relatively abundant lymphaticstructures, contributing to the relapse and metastasis of gastric cancervia the lymphatic system. The lymphatic system is rich in immunecomponents, proper activation of immune cells can reduce the probabilityof relapse and metastasis through killing tumor cells. However, tumorcells often escape from the lysis of immune cells, the mechanisms ofwhich are not completely clear. Therefore it is of significant clinicalvalue to design, on the basis of the tumor immune evasion mechanisms,effective immunotherapeutic strategies, which have become anotherefficient anti-cancer treatment after surgery.

Human immune system can be divided into innate and adaptive components.The adaptive immunity functions through B and T lymphocytes and servesas a secondary response towards specific antigen but usually takeseffect after a period of time. Contrary to it, the innate immunity,including natural killer cells (NK cells), is the first line to defensetumor cells and pathogen. NK cells can react and exert cytotoxicitywithin 4 hours in vitro and in vivo, via release of perforins, NKcytotoxic factors, and tumor necrosis factors. NK cells have been foundto lyse tumor cells in a short amount of time, thus recent emphasis hasbeen made on the development and clinical utilization of NK immunity totreat tumor.

The cytotoxic activity mediated by NK cells is intricately regulated bythe balance between activating signals and suppressive signals. Theproteins of HLA-A, B, and C (i.e., the classic HLA class I molecules)expressed on target cells interact with inhibitory receptors on NK cellsand cause anergy. On the other hand, IL-2 and IL-12 secreted bydendritic cells activate NK cells.

Various remedial practices were devised based on the regulationmechanisms in order to achieve tumor regression by altering thesuppressed status of NK cells, the most renowned ones including usingallogenic NK cells and disrupting the inhibitory receptors on NKsurface. These practices significantly enhanced the anti-cancer effectsof NK-related immune therapies, but they also gave rise to auto-immunesymptoms. Therefore, it remains one of the plausible means of enhancingthe outcome of NK therapies to remold the cancer cells.

SUMMARY OF THE INVENTION

In an aspect, the present invention provides a kit for detecting and/ordiagnosing metastatic potential of cancer cells or for evaluatingprognosis in a patient with cancer comprising a reagent for detecting ina tumor tissue the protein expression level of an HLA class I moleculeand/or the copy number variation of a polynucleotide encoding the HLAclass I molecule, wherein a higher protein expression level of the HLAclass I molecule and/or a copy number amplification of thepolynucleotide encoding the HLA class I molecule compared to that in theadjacent matched normal tissue is indicative of high metastaticpotential of the cancer cells and/or poor prognosis in the patient; anda lower protein expression level of the HLA class I molecule and/or acopy number deletion of the polynucleotide encoding the HLA class Imolecule compared to that in the adjacent matched normal tissue isindicative of low metastatic potential of the cancer cells and/or goodprognosis in the patient.

In an embodiment of this aspect of the present invention, the reagentfor detecting the protein expression level of the HLA class I moleculeand/or the copy number variation of the polynucleotide encoding the HLAclass I molecule is a binding agent that binds to the HLA class Imolecule or a substance that hybridizes with or amplifies thepolynucleotide encoding the HLA class I molecule. In another embodimentof the present invention, binding agent that binds to the HLA class Imolecule is an anti-HLA class I antibody, and the HLA class I moleculeis HLA-A, HLA-B, or HLA-C. Preferably, the HLA class I molecule isHLA-C. In another embodiment of the present invention, the kit furthercomprises a reagent for detecting in the tumor tissue the number orcytotoxic activity of NK cells, wherein a higher number or cytotoxicactivity of NK cells compared to that in the adjacent matched normaltissue indicates low metastatic potential of the cancer cells and/orgood prognosis in the patient. In another embodiment of the presentinvention, the kit further comprises one or more reagents selected fromthe group consisting of: (a) a reagent for detecting in the tumor tissuethe protein expression level of NKp30, (b) a reagent for detecting inthe tumor tissue the protein expression level of pERK, (c) a reagent fordetecting in the tumor tissue the protein expression level of IL-2, and(d) a reagent for detecting in the tumor tissue the protein expressionlevel of IL-12, wherein a lower protein expression level of NKp30, pERK,IL-2, IL-12, or any combination thereof compared to that in the adjacentmatched normal tissue indicates high metastatic potential of the cancercells and/or poor prognosis in the patient. In another embodiment of thepresent invention, the cancer cells are gastric cancer cells, and thecancer is gastric cancer.

A second aspect of the present invention provides a use of a reagent fordetecting in a tumor tissue the protein expression level of an HLA classI molecule and/or the copy number variation of a polynucleotide encodingthe HLA class I molecule in the manufacture of a kit for detectingand/or diagnosing metastatic potential of cancer cells or for evaluatingprognosis in a patient with cancer, wherein a higher protein expressionlevel of the HLA class I molecule and/or a copy number amplification ofthe polynucleotide encoding the HLA class I molecule compared to that inthe adjacent matched normal tissue is indicative of high metastaticpotential of the cancer cells and/or poor prognosis in the patient; anda lower protein expression level of the HLA class I molecule and/or acopy number deletion of the polynucleotide encoding the HLA class Imolecule compared to that in the adjacent matched normal tissue isindicative of low metastatic potential of the cancer cells and/or goodprognosis in the patient.

In an embodiment of this aspect of the present invention, the reagentfor detecting the protein expression level of the HLA class I moleculeand/or the copy number variation of the polynucleotide encoding the HLAclass I molecule is a binding agent that binds to the HLA class Imolecule or a substance that hybridizes with or amplifies thepolynucleotide encoding the HLA class I molecule. In another embodimentof the present invention, the binding agent that binds to the HLA classI molecule is an anti-HLA class I antibody, and the HLA class I moleculeis HLA-A, HLA-B, or HLA-C. In another embodiment of the presentinvention, the reagent for detecting the protein expression level of theHLA class I molecule and/or the copy number variation of thepolynucleotide encoding the HLA class I molecule is combined with areagent for detecting in the tumor tissue the number or cytotoxicactivity of NK cells, wherein a higher number or cytotoxic activity ofNK cells compared to that in the adjacent matched normal tissueindicates low metastatic potential of the cancer cells and/or goodprognosis in the patient. In another embodiment of the presentinvention, the reagent for detecting the protein expression level of theHLA class I molecule and/or the copy number variation of thepolynucleotide encoding the HLA class I molecule is combined with one ormore reagents selected from the group consisting of: (a) a reagent fordetecting in the tumor tissue the protein expression level of NKp30, (b)a reagent for detecting in the tumor tissue the protein expression levelof pERK, (c) a reagent for detecting in the tumor tissue the proteinexpression level of IL-2, and (d) a reagent for detecting in the tumortissue the protein expression level of IL-12, wherein a lower proteinexpression level of NKp30, pERK, IL-2, IL-12, or any combination thereofcompared to that in the adjacent matched normal tissue indicates highmetastatic potential of the cancer cells and/or poor prognosis in thepatient. In another embodiment of the present invention, the cancercells are gastric cancer cells, and the cancer is gastric cancer.

A third aspect of the present invention provides a use of an anti-HLAclass I antibody or an oligonucleotide that down-regulates theexpression of an HLA class I molecule in the manufacture of a medicamentfor enhancing the effect of NK-cell therapy. In an embodiment of thisaspect of the present invention, the medicament further comprises IL-2and/or IL-12. In another embodiment of the present invention, themedicament further comprises a reagent that activates the NKp30/MAPK3signaling pathway. In another embodiment of the present invention, thereagent that activates the NKp30/MAPK3 signaling pathway is anoligonucleotide that targets NKp30 ligand BAG6 and up-regulates theexpression of BAG6 in cancer cells. In another embodiment of the presentinvention, the HLA class I molecule is HLA-A, HLA-B, or HLA-C, and theNK-cell therapy is used for treating cancer. In another embodiment ofthe present invention, the cancer cells express the HLA class Imolecule.

A fourth aspect of the present invention provides a composition fordetecting and/or diagnosing metastatic potential of cancer cells or forevaluating prognosis in a patient with cancer comprising or consistingof one or more reagents selected from the group consisting of: (a) areagent for detecting in a sample the protein expression level of an HLAclass I molecule and/or the copy number variation of a polynucleotideencoding the HLA class I molecule, (b) a reagent for detecting in thesample the number or cytotoxic activity of NK cells, (c) a reagent fordetecting in the sample the protein expression level of IL-2 and/orIL-12, (d) a reagent for detecting in the sample the protein expressionlevel of NKp30, and (e) a reagent for detecting in the sample theprotein expression level of pERK. In an embodiment of this aspect of thepresent invention, the composition comprises: (a) the reagent fordetecting in the sample the protein expression level of an HLA class Imolecule and/or the copy number variation of a polynucleotide encodingthe HLA class I molecule, (b) the reagent for detecting in the samplethe number or cytotoxic activity of NK cells. In another embodiment ofthe present invention, the composition comprises: (a) the reagent fordetecting in the sample the protein expression level of an HLA class Imolecule and/or the copy number variation of a polynucleotide encodingthe HLA class I molecule, and one or more reagents selected from thegroup consisting of (c) the reagent for detecting in the sample theprotein expression level of IL-2 and/or IL-12, (d) the reagent fordetecting in the sample the protein expression level of NKp30, and (e)the reagent for detecting in the sample the protein expression level ofpERK.

A fifth aspect of the present invention provides a kit for assessing thetumorigenicity of cells or cell lines in an individual comprising: (a) areagent for detecting in a sample the protein expression level of an HLAclass I molecule, and (b) a reagent for detecting in the sample thenumber or cytotoxic activity of NK cells, wherein a lower proteinexpression level of the HLA class I molecule and a higher number orcytotoxic activity of NK cells compared to that in a control sample isindicative of no/low tumorigenicity of the cells or cell lines in theindividual.

A sixth aspect of the present invention provides a kit for assessing thetumorigenicity of cells or cell lines in an individual comprising: (a) areagent for detecting in a sample the protein expression level of an HLAclass I molecule, and (b) one or more reagents selected from the groupconsisting of: a reagent for detecting in the sample the proteinexpression level of NKp30, a reagent for detecting in the sample theprotein expression level of pERK, a reagent for detecting in the samplethe protein expression level of IL-2, and a reagent for detecting in thesample the protein expression level of IL-12, wherein a higher proteinexpression level of the HLA class I molecule and a lower proteinexpression level of NKp30, pERK, IL-2, IL-12, or any combination thereofcompared to that in a control sample is indicative of hightumorigenicity of the cells or cell lines in the individual. In anembodiment of this aspect of the present invention, the expression ofNKp30 indicates low tumorigenicity of the cells or cell lines. Inanother embodiment of the present invention, the cells or cell lines aretumor cells of epithelial origin.

In an embodiment of the fifth or sixth aspect of the present invention,the reagent for detecting in a sample the protein expression level of anHLA class I molecule is an anti-HLA class I antibody, and the HLA classI molecule is HLA-A, HLA-B, or HLA-C. In another embodiment of the fifthor sixth aspect of the present invention, the tumor cells are gastriccancer cells. In another embodiment of the fifth or sixth aspect of thepresent invention, the individual is a human being or a nude mouse.

A seventh aspect of the present invention provides a method for in vitroand/or in vivo detecting and/or diagnosing metastatic potential ofcancer cells or for evaluating prognosis in a patient with cancercomprising contacting a cancer cell sample from an individual with areagent for detecting in the cancer cell sample the protein expressionlevel of an HLA class I molecule and/or the copy number variation of apolynucleotide encoding the HLA class I molecule, wherein a higherprotein expression level of the HLA class I molecule and/or a copynumber amplification of the polynucleotide encoding the HLA class Imolecule compared to that in a control sample is indicative of highmetastatic potential of the cancer cells and/or poor prognosis in thepatient. In an embodiment of the present invention, the method furthercomprises contacting the cancer cell sample from the individual with areagent for detecting in the cancer cell sample the number or cytotoxicactivity of NK cells, wherein a higher number or cytotoxic activity ofNK cells compared to that in the control sample indicates low metastaticpotential of the cancer cells and/or good prognosis in the patient. Inanother embodiment of the present invention, the method furthercomprises contacting the cancer cell sample from the individual with oneor more reagents selected from the group consisting of: (a) a reagentfor detecting in the cancer cell sample the protein expression level ofNKp30, (b) a reagent for detecting in the cancer cell sample the proteinexpression level of pERK, (c) a reagent for detecting in the cancer cellsample the protein expression level of IL-2, and (d) a reagent fordetecting in the cancer cell sample the protein expression level ofIL-12, wherein a lower protein expression level of NKp30, pERK, IL-2,IL-12, or any combination thereof compared to that in the control sampleindicates high metastatic potential of the cancer cells and/or poorprognosis in the patient. In another embodiment of the presentinvention, the reagent for detecting the protein expression level of theHLA class I molecule and/or the copy number variation of thepolynucleotide encoding the HLA class I molecule is a binding agent thatbinds to the HLA class I molecule or a substance that hybridizes with oramplifies the polynucleotide encoding the HLA class I molecule. Inanother embodiment of the present invention, the binding agent thatbinds to the HLA class I molecule is an anti-HLA class I antibody, andthe HLA class I molecule is HLA-A, HLA-B, or HLA-C. In anotherembodiment of the present invention, the cancer cells are gastric cancercells, and the cancer is gastric cancer.

An eighth aspect of the present invention provides a use of an HLA classI molecule for detecting and/or diagnosing metastatic potential ofcancer cells or for evaluating prognosis in a patient with cancer. In anembodiment of this aspect of the present invention, the cancer cells aregastric cancer cells, and the cancer is gastric cancer.

An ninth aspect of the present invention provides a use of one or morereagents selected from a group for detecting and/or diagnosingmetastatic potential of cancer cells or for evaluating prognosis in apatient with cancer, wherein the group consists of: (a) a reagent fordetecting in a sample the protein expression level of an HLA class Imolecule and/or the copy number variation of a polynucleotide encodingthe HLA class I molecule, (b) a reagent for detecting in the sample thenumber or cytotoxic activity of NK cells, (c) a reagent for detecting inthe sample the protein expression level of IL-2 and/or IL-12, (d) areagent for detecting in the sample the protein expression level ofNKp30, and (e) a reagent for detecting in the sample the proteinexpression level of pERK. In an embodiment of this aspect of the presentinvention, the reagent for detecting in the sample the proteinexpression level of an HLA class I molecule is an anti-HLA class Iantibody, and the HLA class I molecule is HLA-A, HLA-B, or HLA-C. Inanother embodiment of the present invention, the cancer cells aregastric cancer cells, and the cancer is gastric cancer.

In one of the above aspects of the present invention, the control sampleis the adjacent matched normal tissue, which is away from tumor tissueat least 5 cm. In one of the above aspects of the present invention, thecontrol sample is nontumor cells with no or low expression of HLA classI molecule such as cells at peritumoral areas, or tumor cells with no orlow expression of HLA class I molecule such as the AGS cell line. In anyone of the above aspects of the present invention, the cancer cells ortumor cells are tumor cells of epithelial origin, preferably gastriccancer cells; the cancer is a tumor of epithelial origin, preferablygastric cancer. In another embodiment, the cancer cells or tumor cellsexpress HLA class I molecule.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Tumorigenic capacity of BGC823 and AGS cells in nude mice. Leftpanel, photographs of nude mice bearing tumors from inoculation of2.5×10⁵ BGC823 cells (A), 1×10⁶ BGC823 cells (B), 1×10⁶ AGS cells (C),or 1×10⁷ AGS cells (D). Right panel, kinetic growth of the mice-borntumors derived from inoculation of 2.5×10⁵ BGC823 cells, 1×10⁶ BGC823cells, 1×10⁶ AGS cells, and 1×10⁷ AGS cells.

FIGS. 2A-C. Copy number variations (CNVs) in genes in BGC823 and AGScells. FIG. 2A, CIRCUS diagrams of whole genome sequencing of BGC823 andAGS cells. FIG. 2B, gene copy number of HLA class I molecules in BGC823and AGS cells. FIG. 2C, Real-time PCR detection of HLA-A, HLA-B, andHLA-C relative copy number in BGC823 and AGS cells.

FIG. 3. Diagram of signal pathways involved in NK cytotoxicity and copynumber variations of NK cytotoxicity-related genes in BGC823 and AGScells.

FIGS. 4A-D. Expression of proteins involved in NK cytotoxicity and theirsubcellular localizations in BGC823 and AGS cells. FIGS. 4A and B,immunoblotting of HLA class I molecules (FIG. 4A) and NKp30/VAV2/MAPK3signal pathway (FIG. 4B) in BGC823 and AGS cells. FIGS. 4C and D,immunofluorescence of HLA-A, HLA-B and NKp30 in BGC823 and AGS cells(FIG. 4C), and of HLA-C in such cells before and after stimulation of NKcells derived from nude mice (FIG. 4D).

FIGS. 5A-E. NK cells display cytotoxic activity against AGS but notBGC823 cells. FIG. 5A, cytotoxicity of nude mice NK cells against BGC823and AGS cells in different coculture ratios. FIG. 5B, time-lapse imagingof NK cells lysing of BGC823 and AGS cells. FIG. 5C, Matrigel-coated AGScells are more resistant to NK attack (left) and more tumorigenic innude mice (right). FIGS. 5D and E, tumorigenic capacity of BGC823 (FIG.5D) and AGS (FIG. 5E) cells in NOD/SCID mice that lack NK cells.

FIGS. 6A-E. Critical roles of HLA class I molecules andNKp30/VAV2/MAPK3/IL-12(IL-2) signal pathway in NK-mediated tumor celllysis. FIGS. 6A and B, cytotoxicity assay (FIG. 6A) and time-lapseimaging (FIG. 6B) of NK-tumor coculture in the presence of IL-12 andantibodies against HLA class I molecules. FIG. 6C, immunoblotting ofNKp30/VAV2/MAPK3/IL-12(IL-2) signal pathway in BGC823 and AGS cells.FIG. 6D, immunoblotting of IL-12 in AGS cells under interference ofNKp30 expression. FIG. 6E, time-lapse imaging of NK cytotoxicity againstAGS cells under interference of NKp30 expression or in the presence ofantibodies against IL-12.

FIGS. 7A-C. Expression of classic HLA class I molecules andNKp30/MAPK3/IL-12(IL-2) in various gastric cancer cell lines withdifferent tumorigenic capacity in nude mice. FIGS. 7A and B, real-timePCR detection of copy number variations of HLA class I molecules (FIG.7A) and immunoblotting of HLA class I molecules andNKp30/MAPK3/IL-12(IL-2) (FIG. 7B) in various gastric cancer cell lineswith different tumorigenic capacity in nude mice. FIG. 7C, real-time PCRdetection of copy number variations of HLA class I molecules inmetastatic and non-metastatic tumor tissues.

FIGS. 8A-F. Expression of HLA class I molecules in paired gastric cancersamples. FIGS. 8A and B, immunohistochemical detection of HLA class Imolecules in tumor tissue and adjacent matched normal tissue (FIG. 8A)and in metastatic and non-metastatic tumor tissues (FIG. 8B) of gastriccancer. FIGS. 8C and D, overall survival in patients with low or highHLA class I expression (FIG. 8C) and in patients with low or high HLAclass I combined with high or low NK infiltration (FIG. 8D). FIG. 8E,treatment of tumor in nude mice with anti-HLA class I antibodies reducedthe tumor growth rate. FIG. 8F, hematoxylin-eosin staining of tissues oftreated and control individuals described in FIG. 8E.

DETAILED DESCRIPTION OF THE INVENTION

Several aspects of the invention are described below with reference toexamples for illustration. It should be understood that numerousspecific details, relationships, and methods are set forth to provide afull understanding of the invention. One skilled in the relevant art,however, will readily recognize that the invention can be practicedwithout one or more of the specific details, or with other methods, etc.

The present invention relates to the relationship between the expressionof a newly found cancer marker (e.g., HLA class I molecules) andmetastatic potential of cancer cells or prognosis in patients withcancer. The cancer marker described herein provides means to predict themetastatic potential of cancer cells. Therefore, an embodiment of thepresent invention represents an improvement of cancer markers, and thecancer marker described herein applies to the diagnosis of commoncancers or specific cancers such as gastric cancer. The cancer markerdescribed herein especially applies to the prediction of metastaticpotential of gastric cancer cells and prognosis of patients with gastriccancer. In another embodiment, the newly found cancer marker describedherein (i.e., HLA class I molecules) can be combined with one or more ofthe known cancer markers in the field (such as CEA, NSE, CA 19-9, CA125, CA 72-4, PSA, proGRP, SCC, and NNMT, etc.) or with one or more ofthe other cancer markers described herein (NK cell, NKp30, pERK, IL-2,IL-12 or their combinations), for treatments, or diagnosis of metastaticpotential of cancer cells, or evaluation of prognosis in patients withcancer, or preparation of kits for such uses.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. References to commonterminologies in cellular and molecular biology can be found in Lewin,B., Gene VIII, Science Press (2005), ISBN:978-7-03-014597-0; Dudek, R.W., Cell and molecular biology, Chinacitic Press (2004),ISBN:978-7-50-860075-8; Wang, J., Biochemistry, Higher Education Press(2002), ISBN:978-7-04-011088-3; Kendrew, J. et al., The Encyclopedia ofMolecular Biology, Blackwell Science Ltd. (1994), ISBN 0-632-02182-9;and Meyers, R. A., Molecular Biology and Biotechnology: a ComprehensiveDesk Reference, VCH Publishers, Inc. (1995), ISBN 1-56081-5698. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice of the invention, particularmaterials and methods are described herein.

The term “cancer” refers to all types of cancers or neoplasm ormalignant tumors found in mammals, examples including but not limited tofibrosarcoma, mucous sarcoma, liposarcoma, chondrosarcoma, osteosarcoma,chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendothelial sarcoma, synovialoma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, gastric cancer, colorectal cancer,pancreatic cancer, breast cancer, ovarian cancer, prostatic carcinoma,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma,cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma,Wilms' tumor, cervical cancer, testicular cancer, lung cancer, smallcell lung cancer, bladder cancer, epicytoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma and retinoblastoma. In certain aspects of thepresent invention, the cancer is preferably gastric cancer, morepreferably highly tumorigenic or highly metastatic gastric cancer.

The term “cancer cells” or “tumor cells” refers to a certain type ofmutated cells that initiate cancer or tumor, and are characterized asunregulated growing, transformed, and metastasizing. In certain aspectsof the present invention, the “cancer cells” or “tumor cells” are canceror tumor cells of epithelial origin, preferably gastric cancer cells,more preferably highly tumorigenic or highly metastatic gastric cancercells. In several aspects of the present invention, the cancer or tumorcells express HLA class I molecules such as HLA-A, HLA-B, and HLA-C. Incertain aspects of the present invention, the gastric cancer cells arehighly tumorigenic cells described in the embodiments, such as BGC823,MGC803, SGC7901 and MKN45, etc.

The term “metastatic potential” refers to the ability or possibility ofa cancer cell moving from the initial site to other sites in the body.

The term “sample” means a material known or suspected of expressing orcontaining cancer markers, or binding agents such as antibodies specificfor cancer markers (such as HLA class I molecules). The sample may bederived from a biological source (“biological sample”), such as tissues(e.g. biopsy samples), extracts, or cell cultures, including cells (e.g.tumor cells), cell lysates, and biological or physiological fluids, suchas, for example, whole blood, plasma, serum, saliva, cerebral spinalfluid, sweat, urine, milk, peritoneal fluid and the like. A sample maybe used directly as obtained from the source or following a pretreatmentto modify the character of the sample, such as preparing plasma fromblood, diluting viscous fluid, and the like. In certain aspects of theinvention, the sample is a human physiological fluid, such as humanserum. In certain aspects of the invention, the sample is a biopsysample, such as tumor tissues or cells obtained from biopsy. In certainaspects of the invention, the sample is a malignant or normal tissuesample, such as peritumoral normal tissues or adjacent matched normaltissues.

Samples that may be analyzed in accordance with the invention includepolynucleotides from clinically relevant sources. As will be appreciatedby those skilled in the art, the target polynucleotides can compriseRNA, including but not limited to total cellular RNA, poly(A)⁺ messengerRNA (mRNA) or fraction thereof, cytoplasmic mRNA, or RNA transcribedfrom cDNA (i.e., cRNA).

Target polynucleotides can be detectably labeled at one or morenucleotides using methods known in the art. The detectable label can be,without limitation, a luminescent label, fluorescent label, biologicalluminescent label, chemical luminescent label, radiolabel, andcolorimetric label.

The term “marker” as used herein refers to a molecule to be used as atarget for analyzing a patient's test sample. Examples of such moleculartargets are proteins or polypeptides. Proteins or polypeptides used as amarker in the present invention are contemplated to include naturallyoccurring variants of said protein as well as fragments of said proteinor said variant, in particular, immunologically detectable fragments.Immunologically detectable fragments preferably comprise at least 6, 7,8, 10, 12, 15 or 20 contiguous amino acids of said marker polypeptide.One of skill in the art would recognize that proteins which are releasedby cells or present in the extracellular matrix may be damaged, e.g.,during inflammation, and could become degraded or cleaved into suchfragments. Certain markers are synthesized in an inactive form, whichmay be subsequently activated by proteolysis. As the skilled artisanwill appreciate, proteins or fragments thereof may also be present aspart of a complex. Such complex also may be used as a marker in thesense of the present invention. Variants of a marker polypeptide areencoded by the same gene, but may differ in their isoelectric point(=PI) or molecular weight (=MW), or both e.g., as a result ofalternative mRNA or pre-mRNA processing. The amino acid sequence of avariant is to 95% or more identical to the corresponding markersequence. In addition, or in the alternative a marker polypeptide or avariant thereof may carry a post-translational modification.Non-limiting examples for posttranslational modifications areglycosylation, acylation, and/or phosphorylation.

The expression of the marker can be identified by detection of markertranslation (i.e., detection of marker protein in a sample). Methodssuitable for the detection of marker protein include any suitable methodfor detecting and/or measuring proteins from a cell or cell extract.Such methods include, but are not limited to, immunoblot (e.g., Westernblot), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), immunoprecipitation, immunohistochemistry and immunofluorescence.Particularly preferred methods for detection of proteins include anycell based assay, including immunohistochemistry and immunofluorescenceassays. Such methods are well known in the art.

The terms “subject”, “patient” and “individual” are interchangeablyherein and refer to a warm-blooded animal such as a mammal that isafflicted with, or suspected of having, being pre-disposed to, or beingscreened for cancer, in particular cancer with metastatic potential. Theterm includes but is not limited to domestic animals, rodents (such asrats and mice), primates and humans. Preferably, the term refers to ahuman.

The term “treating”, “treatment” or “therapy” covers the treatment of adisease-state in a mammal, and includes: (a) preventing thedisease-state from occurring in a mammal, in particular, when suchmammal is predisposed to the disease-state but has not yet beendiagnosed as having it; (b) inhibiting the disease-state, e.g.,arresting it development; and/or (c) relieving the disease-state, e.g.,causing regression of the disease state until a desired endpoint isreached. Treating also includes the amelioration of a symptom of adisease (e.g., lessen the pain or discomfort), wherein such ameliorationmay or may not be directly affecting the disease (e.g., cause,transmission, expression, etc.).

The terms “polypeptide” and “protein” are used interchangeably hereinand indicate at least one molecular chain of amino acids linked throughcovalent and/or non-covalent bonds. The terms include peptides,oligopeptides, and proteins, and post-translational modifications of thepolypeptides, e.g. glycosylations, acetylations, phosphorylations, andthe like. Protein fragments, analogues, mutated or variant proteins,fusion proteins, and the like, are also included within the meaning ofthe terms.

In certain embodiments of the present invention, detection of “proteinexpression levels”, “gene expression”, or “gene expression levels”includes but is not limited to detection of corresponding RNA, protein,or polypeptide levels (or their combinations). Specific methods andreagents for the detection of protein, polypeptide, or RNA levels arenot restricted in the present invention, and are well known in thefield.

Methods for measuring in a sample the quantity or concentration of aprotein is known by those skilled in the art. These methods include RIA,competitive binding assay, protein blotting assay, and ELISA. In methodsthat involve usage of antibodies, both monoclonal and polyclonalantibodies are applicable, and the antibodies are immunologicallyspecific against the protein, the epitope or the fragment.

The term “polynucleotide” refers to a polymeric form of nucleotides ofany length, either ribonucleotides or deoxyribonucleotides. The termincludes double- and single-stranded DNA and RNA, modifications such asmethylation or capping and unmodified forms of the polynucleotide. Theterms “polynucleotide” and “oligonucleotide” are used interchangeablyherein. A polynucleotide may, but need not, include additional coding ornon-coding sequences, or it may, but need not, be linked to othermolecules and/or carrier or support materials. Polynucleotides for usein the methods or kit of the invention may be of any length suitable fora particular method or kit. In certain applications the term refers toantisense nucleic acid molecules (e.g. an mRNA or DNA strand in thereverse orientation to a sense of polynucleotides encoding cancermarkers of the present invention such as HLA class I molecules).

The polynucleotide cancer markers in the present invention includepolynucleotides encoding polypeptide cancer markers (for example, HLAclass I molecules), including a native-sequence polypeptide, apolypeptide variant including a portion of the polypeptide cancermarker, an isoform, a precursor, and a chimeric polypeptide. Apolynucleotide encoding an HLA class I polypeptide that can be employedin the present invention includes but is not limited to nucleic acidscomprising a sequence of GenBank Accession Nos. Z46633, D83043, orD83957 or fragments thereof.

Polynucleotides used in the methods of the invention includecomplementary nucleic acid sequences and nucleic acids that aresubstantially identical to these sequences. The polynucleotides alsoinclude sequences that differ from a nucleic acid sequence due todegeneracy in the genetic code. Polynucleotides used in the methods ofthe present invention may also include nucleic acids that hybridizeunder stringent conditions, preferably high stringency conditions to anucleic acid sequence of a polynucleotide cancer marker.

Polynucleotide hybridization assays are well known in the art,Hybridization assay procedures and conditions will vary depending on theapplication and are selected in accordance with the general bindingmethods known including those referred to in: Sambrook, J., et al., TheCondensed Protocols From Molecular Cloning: A Laboratory Manual, SciencePress (2002), ISBN:7-03-010338-6; Young and Davis, Proc. Natl. Acad.Sci. USA 80:1194 (1983). Methods and apparatus for carrying out repeatedand controlled hybridization reactions have been described in U.S. Pat.Nos. 5,871,928, 5,874,219, 6,045,996, 6,386,749 and 6,391,623 each ofwhich are incorporated herein by reference.

Under certain circumstances, the sample may need amplification. Agenomic sample may be amplified by a variety of mechanisms, some ofwhich may employ PCR. The sample may be amplified on the array. See, forexample, U.S. Pat. No. 6,300,070 and U.S. patent application Ser. No.09/513,300, which are incorporated herein by reference.

Other suitable amplification methods include the ligase chain reaction(LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al,Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad, Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al, Proc. Nat. Acad. Sci, USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 5,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517 and 6,063,603, each of which is incorporatedherein by reference).

A “higher” or “lower” marker expression level or a copy number“amplification” or “deletion” of a polynucleotide encoding the marker ina sample of a patient compared to that in a control or standard (e.g., anormal level, levels in different stages, or levels in other samples ofthe patient) indicates that the levels in the sample are at least about1.25, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more higher, or atmost about 1/1.25, 1/1.5, 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, or1/10 or less, respectively, than the control or standard. The copynumber amplification or deletion may be detected by techniques wellknown in the field, such as the whole genome sequencing described in theexamples.

Reagents for detecting the protein expression level of an HLA class Imolecule and/or the copy number variation of a polynucleotide encodingthe HLA class I molecule are well known in the field. Such reagents thatcan be employed in the present invention may be commercially availableor prepared through methods well appreciated by those skilled in theart. For instance, in an embodiment of the present invention, suchreagent is a binding agent that binds to the HLA class I molecule or asubstance that hybridizes with or amplifies the polynucleotide encodingthe HLA class I molecule.

The term “binding agent” refers to a substance such as a polypeptide,antibody, ribosome, or aptamer that specifically binds to a cancermarker of the present invention (such as an HLA class I molecule). Asubstance “specifically binds” to a polypeptide cancer marker in theinvention if it reacts at a detectable level with the polypeptide cancermarker, and does not react detectably with peptides containing unrelatedsequences or sequences of different polypeptides. Binding properties maybe assessed by ELISA, which may be readily performed by those skilled inthe art.

A binding agent may be a ribosome, with or without a peptide component,an RNA or DNA molecule, or a polypeptide. A binding agent may be apolypeptide that comprises a polypeptide sequence of a cancer marker HLAclass I, a peptide variant thereof, or a non-peptide mimetic of such asequence.

An aptamer includes a DNA or RNA molecule that binds to nucleic acidsand proteins. An aptamer that binds to a cancer marker of the presentinvention can be produced using conventional techniques, without undueexperimentation. For example, see the following publications describingin vitro selection of aptamers: Mug et al., Mol. Biol. Reports 20:97-107(1994); Wallis et al., Chem. Biol. 2:543-552 (1995); Ellington, Curr.Biol. 4:427-429 (1994); Lato et al., Chem. Biol. 2:291-303 (1995);Conrad et al., Mol. Div. 1:69-78 (1995); and Uphoff et al., Curr. Opin.Struct. Biol. 6:281-287 (1996).

Antibodies for use in the present invention include but are not limitedto synthetic antibodies, monoclonal antibodies, polyclonal antibodies,recombinant antibodies, antibody fragments (such as Fab, Fab′, F(ab′)₂),dAb (domain antibody; see Ward et al., 1989, Nature 341:544-546),antibody heavy chains, intrabodies, humanized antibodies, humanantibodies, antibody light chains, single chain Fvs (scFv) (e.g.,including monospecific, bispecific, etc.), anti-idiotypic (ant-Id)antibodies, proteins comprising an antibody portion, chimeric antibodies(for example, antibodies which contain the binding specificity of murineantibodies, but in which the remaining portions are of human origin),derivatives, such as enzyme conjugates or labelled derivatives,diabodies, linear antibodies, disulfide-linked Fvs (sdFv), multispecificantibodies (e.g., bispecific antibodies), epitope-binding fragments ofany of the above, and any other modified configuration of animmunoglobulin molecule that comprises an antigen recognition site ofthe required specificity. An antibody includes an antibody of any type(e.g. IgA, IgD, IgE, IgG, IgM and IgY), any class (e.g. IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2), or any subclass (e.g. IgG2a and IgG2b), andthe antibody need not be of any particular type, class or subclass. Incertain embodiments of the invention the antibodies are IgG antibodiesor a class or subclass thereof. An antibody may be from any animalorigin including birds and mammals (e.g. human, murine, donkey, sheep,rabbit, goat, guinea pig, camel, horse, or chicken).

By way of example, antibodies used in the invention may be purchasedfrom suppliers such as Santa Cruz Biotechnology (anti-HLA class Iantibody, B425 (246-B8.E7), catalog No. sc-59204), ProteinTech Group(anti-HLA-A antibody, catalog Nos. 66013-1-Ig and 15240-1-AP; anti-HLA-Bantibody, catalog No. 17260-1-AP; anti-HLA-C antibody, catalog No.15777-1-AP), Abcam (e.g. antibodies of catalog Nos. ab33252 andab79523), Epitomics (antibodies of catalog Nos. 1913-1, 2389-1, and5472-1), and the like. Otherwise, the antibodies may be prepared byrecombinant methods well known in the field. In certain embodiments, theantibodies are monoclonal antibodies. See, for example, Kohler et al.,Nature 256:495-497 (1975); Kozbor et al., J. Immunol. Methods 81:31-42(1985); Cote et al., Proc. Natl. Acad. Sci. USA 80:2026-2030 (1983); andCole et al., Mol. Cell Biol. 62: 109-120 (1984) for the preparation ofmonoclonal antibodies.

In certain embodiments of the invention, reagents for detecting theprotein expression level of an HLA class I molecule and/or the copynumber variation of a polynucleotide encoding the HLA class I moleculemay be obtained through methods and systems described in CN1703624A. Insome other embodiments, the reagents are peptides/antibodies orpolynucleotides identified in CN101287755A, WO2012176879, or WO2011037160, or prepared by methods described in CN101287755A,WO2012176879, or WO 2011037160.

Reagents that may be used for detecting the number or cytotoxic activityof NK cells are well known in the field. Such reagents applicable in thepresent invention may be purchased or routinely prepared through methodswell known by those skilled in the art. By way of example, such reagentsmay be those used in NK cell number detection by flow cytometry, thoseused in NK cytotoxic activity examination by MTT colorimetric assay(see, for example, Mosmann T., 1983, J. Immunol. Methods 65:55), LDHdetermination method (see, for example U.S. Pat. No. 4,006,061 A), and⁵¹Cr release assay (see, for example, Mariana M. Mata., 2014, J.Immunol. Methods 406:1-9).

Likewise, reagents for detecting NKp30, pERK, IL-2 and IL-12 are wellknown in the field. Such reagents applicable in the present inventionmay be purchased or routinely prepared through methods well known bythose skilled in the art.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not of limitation. Those of skill in the art should,in light of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention. All references, including publications, patentapplications, and patents, cited herein are incorporated by reference intheir entirety and to the same extent as if each reference wereindividually and specifically indicated to be incorporated by referenceand were set forth in its entirety herein.

EXAMPLES

Unless specified otherwise, all materials used in the examples werecommercially available and were of analytical grade at least, and allspecific experimental procedures employed were conventional methods inthe field (see, for example, Ausubel, E M., et al., Short Protocols inMolecular Biology, Science Press (1999), ISBN:7-03-006408-9; andSambrook, J., et al., The Condensed Protocols From Molecular Cloning: ALaboratory Manual, Science Press (2002), ISBN:7-03-010338-6), and may bedetermined routinely under necessity by those skilled in the art.Details in certain materials and methods are described below.

Materials and Methods 1. Antibodies

Anti-HLA-A antibody (1913-1; Epitomics), anti-HLA-B antibody (2389-1;Epitomics), anti-HLA-C antibody (5472-1; Epitomics), anti-MICA antibody(T3305; Epitomics), anti-VAV2 antibody (B1241; anbo), anti-MAPK3antibody (C11133; anbo), anti-NKp30 antibody (BS3888; Bioworld),anti-pERK antibody (Tyr204) (sc-7383; Santa Cruz), anti-BAG6 antibody(6763; Epitomics), anti-actin antibody (A5441; Sigma).

2. Cell Lines

All cell lines were purchased from ATCC or the cell bank of XieheHospital, Beijing.

3. Quantitative Real-Time PCR (qPCR)

Primers:

Gene Forward (5′------3′) Reverse (5′------3′) LINE-1 AAAGCCGCTCAACTACATGG TGCTTTGAATGCGTCCCAGAG HLA-A GTAAGGAGGGAGATGGGGGTCAGCAATGATGCCCACGATG HLA-B TGAGATGGGGTAAGGAGGGG CACAACTGCTAGGACAGCCAHLA-C GTCCAGAACCCACAACTGCT TGCCAGAGGCTCTTGAAGTC

The qPCR experiments were performed using cDNA of gastric cancer celllines or paired gastric cancer samples. Data obtained were analyzed by2^(−ΔΔct) interpretation through ABI SDS Software, using LINE-1 asreference.

4. Whole Genome Sequencing (WGS)

WGS of paired-end whole-genome shotgun reads were obtained. Reads werealigned to the reference genome (National Center for BiotechnologyInformation Build 37) with Burrows-Wheeler Aligner. The copy numbervariations (CNVs) were predicted by ReadDepth (C. A. Miller, O. Hampton,C. Coarfa, A. Milosavljevic, 2011, ReadDepth: a parallel R package fordetecting copy number alterations from short sequencing reads. PloS one6, e16327). Two copies of chromosome 4 were suggested in the two celllines when the assessment was accomplished by karyotype and sequencingdepth analysis. Obtained fragment ratios were normalized by the copynumber of chromosome 4. The CNV was identified as amplification at log₂ratio >0.45 and deletion at log 2 ratio <0.45, respectively.

5. Immunohistochemistry

Paraffin sections were deparaffinized in xylene twice, 30 min each, andthen were transferred to 100%, 95%, 90%, 85%, and 80% alcoholrespectively for 5 min each for rehydration. After rinsed in ddH₂Othrice, the slides were submerged in 10 mM citrate buffer (pH 6.0) andantigen retrieval was performed with microwave heating. After rinsed inddH₂O thrice, the sections were incubated in 3% H₂O₂ solution at roomtemperature for 10 min to block endogenous peroxidase activity. Afterrinsed in PBS twice, sections were blocked with 5% milk for 30 min atroom temperature. Appropriately diluted primary antibodies were added tothe PBS-rinsed sections and the slides were incubated in a humidifiedchamber at 4° C. overnight and were washed in PBS thrice. Appropriatelydiluted biotinylated secondary antibodies were added to the sections andthe slides were incubated in a humidified chamber at room temperaturefor 30 min and were washed in PBS thrice. Appropriately dilutedStreptavidin-HRP conjugates were added to the sections and the slideswere incubated in a humidified chamber at room temperature for 30 min inthe dark. DAB substrate solution was applied after the sections werewashed in PBS thrice and the color of antibody staining was revealed.The reaction was terminated in PBS and sections were washed in tapwater. Slides were counterstained by Hematoxylin and decolorized inhydrochloric acid-alcohol, and then blued in weak alkaline water. Theslides were then dehydrated through 80%, 85%, 90%, 95%, and 100%alcohol, cleared in xylene for 5 min, and mounted with resinene.Staining was observed under microscopy.

6. Statistical Analyses

Data were subjected to statistical analysis using SPSS 16.0 software(Chicago). Gene expression was compared in different samples usingChi-square (X²) test and t-test. Prognostic analyses were performed withKaplan-Meier methods and Cox multivariate model. P<0.05 was consideredstatistically significant.

7. Animal Model

Several human tumor cell lines are tumorigenic in nude mice that lackthymus and T cell immunity. Still, some xenografts are not able to formtumor mass in nude mice, which we speculate is due to host NK activity.Clinical studies have shown that a proportion of cancer patients are notresponsive to NK transfusion therapies, the mechanism of which remainsunclear. To address this problem, here in the invention we used nudemice as animal models, the highly tumorigenic gastric cell line BGC823and the non-tumorigenic AGS as tumor cell models, and investigated themechanisms by which tumor cells evade NK cytotoxicity. The goal wasachieved by comparing the genome sequencing data of the two cell linesthereby identifying the key genetic factors that caused AGS cells beingtargeted for and BGC823 escaping NK-mediated cytolysis. We examined theclinical significance of these key factors in mass samples andpreliminarily explored their clinical application.

Example 1 Tumor Formation in Nude Mice

In order to examine the relationship between tumor cell linetumorigenicity in nude mice and NK cell activity, and to study themechanism by which tumor cells evade NK cytotoxicity, selection of tumorcell lines with high tumorigenicity and non-tumorigenicity in nude micewas a necessary approach. We evaluated the tumorigenicity of tumor celllines by subcutaneous injection of different numbers of cells of thecell lines. As shown in FIG. 1 and Table 1, cell line BGC823 proved tobe highly tumorigenic in that 100% tumor incidence was observed on Day11 when the injected cell number was 2.5×10⁵; AGS cells, on the otherhand, failed to form a tumor mass in 120 days after injection withinjected cell numbers up to 1×10⁷. Accordingly, we chose the highlytumorigenic BGC823 cell line and the non-tumorigenic AGS cell line asobjects of study.

TABLE 1 Tumor formation of BGC823 and AGS cells in nude mice injectedCell number Cell line 0.25 × 10⁶ 0.3 × 10⁶ 0.5 × 10⁶ 1 × 10⁶ 5 × 10⁶ 1 ×10⁷ BGC823 4/4 63/66 18/19 4/4 — — (11 d) (10 d) (7 d) (3 d) AGS — — —0/3 0/11 0/10 (90 d) (120 d) (120 d)

Example 2 Analysis of CNV Between BGC823 and AGS and Identification ofNK Cytotoxicity-Related Genes

Previous studies have shown that gene mutations and chromosome anomaliesare key events in tumor pathogenesis. Many studies showed thatvariations in gene copy number were more constant than gene mutations inthe onset and development of cancer. Thus we examined the copy numbervariations between the two cell lines by whole genome sequencing. FIG.2A showed great disparity between copy numbers of the two cell lines:BGC823 was triploid while AGS was diploid. NK cells are known to targetcells with no or low expressions of HLA class I molecules (mainly HLA-A,HLA-B and HLA-C), hence we specifically analyzed the HLA class I regionon Chromosome 6 of the two cell lines. As shown in FIG. 2B, there wasevident amplification of the region in the BGC823 genome, while deletionof HLA-C was observed in AGS. This result was confirmed by real-time PCR(FIG. 2C) which revealed significantly higher expression of HLA class Imolecules in BGC823 cells than in AGS cells.

To identify more NK cytotoxicity-related genes, we conducted a KEGGanalysis on genes with CNV and found a series of related genes (FIG. 3):HLA class I molecules are expressed on the surface of target cells andinteract with inhibitory receptors (killer-cell immunoglobulin-likereceptors, KIRs) on NK cells, thereby suppress NK cytotoxicity againstthe target tumor cells. Ligand BAG6 is expressed by target cells, BAG6can activate NKp30 on NK cells and the downstream VAV2/MAPK3 pathway,which leads to interferon production by NK cells and subsequentcytotoxicity against target cells.

Example 3 Examination of Protein Expression of the NKCytotoxicity-Related Genes with CNV

As proteins are the ultimate executors of gene functions, we examinedthe levels of the protein products of the above mentionedcytotoxicity-related genes that with CNV, and established theirsubcellular localizations by immunofluorescence. As shown in FIG. 4A,the protein expressions of HLA class I molecules were evidently higherin BGC823 than in AGS cells, which is consistent with the copy numberdata. The fluorescence signals revealed membrane distribution patternsof HLA-A and HLA-B in BGC823 cells but only weak expression of theseproteins in AGS cells (FIG. 4C). Upon stimulation from nude mice NKcells, HLA-C molecules in BGC823 cells translocated from cytoplasm tocell membrane, while in AGS cells, no such molecule was detected beforeand after NK stimulation, and AGS cells displayed apoptotic morphologyafter NK contact, suggesting active NK cytotoxicity against these AGScells (FIG. 4D).

In accordance with the KEGG analysis, we subsequently detected theprotein expression of other NK cytotoxicity-related genes (FIG. 4B).NKp30 was reported to be expressed only on NK cells in previous studies.Our results, however, showed evident expression of this molecule on thecell membrane of AGS cells which are non-tumorigenic in nude mice (FIGS.4B-C). Also in AGS cells, higher levels of NKp30 downstream VAV2 andMAPK3 was detected, compared with those in BGC823 cells, suggestingexistence of NK activation pathways inside non-tumorigenic tumor cells,which might be the cause of these cells being susceptible to NK attacks.

Example 4 Examination of Nude Mice NK Cytotoxicity Against HighlyTumorigenic BGC823 Cells and Non-Tumorigenic AGS Cells

The results above have established the following phenomena: expressionof HLA class I genes that inhibit NK activity is elevated in highlytumorigenic BGC823 cells but down-regulated in non-tumorigenic AGScells; NK activation pathways are inactive in highly tumorigenic BGC823cells but active in non-tumorigenic AGS cells; following nude mice NKcell stimulation, apoptotic bodies can be observed in AGS cells. Theseevidences suggest that NK cytotoxicity against AGS cells might be thecause of the non-tumorigenicity of AGS cells in nude mice. Todemonstrate this hypothesis, we isolated NK cells from nude mice andcocultured them with BGC823 and AGS cells. Cytotoxicity assays showedthat NK cells lysed AGS cells and that this was NK-cell numberdependent; however, NK cells did not react to BGC823 cells (FIG. 5A).Using time-lapse imaging, we observed that NK cells recognized andattacked AGS cells as soon as they were mixed together, but they had nodetectable response towards BGC823 cells (FIG. 5B). We subsequentlycoated AGS cells with Matrigel to separate them from NK cells and thenconducted cytotoxicity assays and tumor formation in nude mice. Underthese conditions, the extent of lysis for AGS cells was lower comparedwith uncoated AGS cells; AGS cells coated with Matrigel even acquiredtumorigenic capacity in nude mice (FIG. 5C). To further confirm thehypothesis, NOD/SCID mice, which lacked NK immunity, were injected withBGC823 or AGS cells. As shown in 100% tumor incidence was observed forboth cell lines in NOD/SCID mice. These results reveal that cytotoxicityof NK cells resulted in AGS cells failing to form tumors in nude mice.

Example 5 High Expression of HLA Class I in Tumor Cells Fosters Evasionof NK Cytotoxicity

To clarify the mechanism of NK cells lysing AGS cells but not BGC823cells, the role of HLA class I molecules was studied first. NKcytotoxicity assays were conducted under blocking of HLA-A, HLA-B andHLA-C by specific antibodies, and as shown in FIG. 6A, NK cells wereable to lyse BGC823 cells in the presence of anti-HLA class I antibodiesand recombinant NK-activating cytokine IL-12. For AGS cells, there waslittle difference between the control and treatment groups because ofthe low expression of HLA class I. NK's cytolytic effects on BGC823cells following incubation with IL-12 and antibodies against HLA class Iwas further confirmed by time-lapse imaging (FIG. 6B).

Example 6 Activation of NKp30/VAV2/MAPK3/IL-12 Pathway Promotes NKCytotoxicity Against Tumor Cells

There are many types of somatic cells, such as erythrocytes, expressedlow levels of HLA molecules on the surface but will not be targeted byNK cells, which suggests activating mechanisms apart from HLA inhibitorysignals in the regulation of NK activity. In immune cells, activation ofNKp30/VAV2/MAPK3/IL-12(IL-2) pathway may lead to IL-12/IL-2 production,which may activate NK cells. Previous studies reported expression ofNKp30 only on the surface of immune cells, but we found NKp30 expressedon the cell membrane of the non-tumorigenic AGS cells, though not onBGC823 cells which are highly tumorigenic (FIG. 4C). We also found thatNKp30 ligand BAG6 was expressed in AGS cells but not in BGC823 cells(FIG. 6C). Levels of downstream molecules of NKp30 signals pERK andIL-12 were also significantly higher in AGS cells than in BGC823 cells(FIG. 6C). Moreover, IL-12 in AGS was down-regulated when NKp30expression was interfered (FIG. 6D). Time-lapse imaging showed that NKcells did not lyse AGS cells when expression of NKp30 was knocked down,or when IL-12 was blocked with a specific antibody (FIG. 6E). These dataindicate that AGS cells up-regulate IL-12 production by activation ofNKp30/MAPK3 pathway, which in turn promotes NK cytotoxicity against AGScells; this pathway is necessary in NK-mediated tumor cell lysing.

Example 7 Expression of Classic HLA Class I Family andNKp30/MAPK3/IL-12(IL-2) in Other Gastric Cancer Cells with DifferentTumorigenic Capacity

To examine whether the expression pattern of classic HLA class I andNKp30 also existed in other gastric cancer cell lines with differenttumorigenic capacity, five other gastric cancer cell lines wereinvestigated. The cell lines MGC803, SGC7901, and MKN45 exhibited hightumorigenicity similar to BGC823 cells, while N87 and KATOIII cells,similar to AGS cells, exhibited low tumorigenicity in nude mice.Real-time PCR was employed to determine CNV of HLA-A, HLA-B and HLA-C inthese cell lines, and as shown in FIG. 7A, higher copy numbers of HLAclass I were seen in cell lines with high tumorigenicity. Immunoblottingwas then used to detect the protein levels of HLA class I andNKp30/MAPK3/IL-12(IL-2) pathway, which confirmed the finding in the qPCRexperiment (FIG. 7B). In contrast, protein levels of BAG6, NKp30, pERK,IL-12, and IL-2 were higher in cells with low tumorigenicity (FIG. 7B).These results suggest that the copy numbers of HLA class I molecules, orthe protein expression levels of HLA class I molecules andNKp30/MAPK3/IL-12(IL-2) pathway may be used for predicting thetumorigenic capacity of cancer cell lines in nude mice.

Example 8 CNVs and Expression Levels of Classic HLA Class I Molecules inPaired Gastric Cancer Tissues

To assess the clinical significance of HLA class I, we conductedimmunohistochemical analysis of tumor tissues and the adjacent matchednormal tissues from 100 cases of gastric cancer. We found that theexpression of HLA-A, HLA-B and HLA-C was significantly higher in thetumor tissue than in the adjacent matched normal tissue area (FIG. 8A;Table 2), which suggests an association between HLA class I expressionand tumorigenesis. Further analysis revealed that lowly expressedclassic HLA class I was associated with non-metastasis (both lymph nodeand distant; P=0.005; Table 3; FIG. 8B), as studies in cell lines haveshown high consistency between the copy number and the proteinexpression of HLA class I. Thus we divided the patient samples intometastatic and non-metastatic groups and examined the CNV of HLA classI. As shown in FIG. 7C, HLA class I copy numbers in non-metastatic tumortissues were lower than in nontumor tissues of the group, while in themetastatic group tumor tissues contained higher copy numbers of HLAclass I, suggesting that HLA class I copy number may be used forpredicting the metastatic potential of gastric cancer. Survival analysisshowed that highly expressed classic HLA class I was an independentpredictor for poor prognosis (P=0.008, HR=2.758, 95% CI=1.3-5.8; FIG.8C). We also examined the NK infiltration in these samples, and foundthat a combination of NK infiltration with lowly expressed classic HLAclass I exhibited a higher degree of association with non-metastasisthan lowly expressed classic HLA class I alone (P<0.001 vs P=0.005;Table 3). Furthermore, we found that combining NK cells infiltrationwith classic HLA class I expression performed even better in predictingpatient prognosis (FIG. 8D).

TABLE 2 Immunohistochemical analysis of HLA class I expression ingastric cancer tissues HLA class I expression Total case HistologyNegative Positive number P value Nontumor 62 (62.0%) 38 (38.0%) 1000.001 Tumor 38 (38.0%) 62 (62.0%) 100

TABLE 3 Association between metastasis and histological features ingastric cancer patients Metastasis Total case Feature − + number P valueHLA class I Positive  9 (14.5%) 53 (85.5%) 62 0.005 expression Negative15 (39.5%) 23 (60.5%) 38 Low HLA plus Yes 14 (50.0%) 14 (50.0%) 28<0.001 NK infiltration No 10 (13.9%) 62 (86.1%) 72

Example 9 Antagonizing HLA Class I May Enhance the Effect of NKImmunotherapy

Above data suggested that targeting classic HLA class I expression mightenhance the effect of NK therapy. To test this hypothesis, we treatedtumor-bearing mice with IL-12 and treated tumor with antibody againstHLA class I (treatment group) or IgG (mock group). Compared withcontrol, a drastic inhibition of tumor growth was observed in treatmentgroup (FIG. 8E). Hematoxylin and eosin (HE) staining of tissue inresponsive individuals showed that immune cell infiltrated andsurrounded tumor cells (FIG. 8F). This result indicates that combinedtreatment of IL-12 and antibody against HLA class I enhance NKimmunotherapy.

1. A method for detecting and/or diagnosing metastatic potential ofcancer cells or for evaluating prognosis in a patient with cancercomprising detecting in a tumor tissue the protein expression level ofan HLA class I molecule and/or the copy number variation of apolynucleotide encoding the HLA class I molecule, wherein a higherprotein expression level of the HLA class I molecule and/or a copynumber amplification of the polynucleotide encoding the HLA class Imolecule compared to that in the adjacent matched normal tissue isindicative of high metastatic potential of the cancer cells and/or poorprognosis in the patient; and a lower protein expression level of theHLA class I molecule and/or a copy number deletion of the polynucleotideencoding the HLA class I molecule compared to that in the adjacentmatched normal tissue is indicative of low metastatic potential of thecancer cells and/or good prognosis in the patient.
 2. The method ofclaim 1, wherein the protein expression level of the HLA class Imolecule and/or the copy number variation of the polynucleotide encodingthe HLA class I molecule is detected with a binding agent that binds tothe HLA class I molecule or a substance that hybridizes with oramplifies the polynucleotide encoding the HLA class I molecule.
 3. Themethod of claim 2, wherein the binding agent that binds to the HLA classI molecule is an anti-HLA class I antibody, and the HLA class I moleculeis HLA-A, HLA-B, or HLA-C.
 4. The method of claim 1, further comprisingdetecting in the tumor tissue the number or cytotoxic activity of NKcells, wherein a higher number or cytotoxic activity of NK cellscompared to that in the adjacent matched normal tissue indicates lowmetastatic potential of the cancer cells and/or good prognosis in thepatient.
 5. The method of claim 1, further comprising one or more stepsselected from the group consisting of: (a) detecting in the tumor tissuethe protein expression level of NKp30, (b) detecting in the tumor tissuethe protein expression level of pERK, (c) detecting in the tumor tissuethe protein expression level of IL-2, and (d) detecting in the tumortissue the protein expression level of IL-12, wherein a lower proteinexpression level of NKp30, pERK, IL-2, IL-12, or any combination thereofcompared to that in the adjacent matched normal tissue indicates highmetastatic potential of the cancer cells and/or poor prognosis in thepatient.
 6. The method of claim 1, wherein the cancer cells are gastriccancer cells, and the cancer is gastric cancer.
 7. A method forenhancing NK-cell therapy, comprising administering to a patientreceiving NK-cell therapy an anti-HLA class I antibody or anoligonucleotide that down-regulates the expression of an HLA class Imolecule thereby enhancing the effect of NK-cell therapy.
 8. The methodof claim 7, wherein the patient is also administered IL-2 and/or IL-12.9. The method of claim 7, wherein the patient is also administered areagent that activates the NKp30/MAPK3 signaling pathway.
 10. The methodof claim 9, wherein the reagent that activates the NKp30/MAPK3 signalingpathway is an oligonucleotide that targets NKp30 ligand BAG6 andup-regulates the expression of BAG6 in cancer cells.
 11. The method ofclaim 7, wherein the HLA class I molecule is HLA-A, HLA-B, or HLA-C, andthe NK-cell therapy is used for treating cancer in the patient.
 12. Acomposition for detecting and/or diagnosing metastatic potential ofcancer cells or for evaluating prognosis in a patient with cancercomprising or consisting of one or more reagents selected from the groupconsisting of: (a) a reagent for detecting in a sample the proteinexpression level of an HLA class I molecule and/or the copy numbervariation of a polynucleotide encoding the HLA class I molecule, (b) areagent for detecting in the sample the number or cytotoxic activity ofNK cells, (c) a reagent for detecting in the sample the proteinexpression level of IL-2 and/or IL-12, (d) a reagent for detecting inthe sample the protein expression level of NKp30, and (e) a reagent fordetecting in the sample the protein expression level of pERK.
 13. A kitfor assessing the tumorigenicity of cells or cell lines in an individualcomprising: (a) a reagent for detecting in a sample the proteinexpression level of an HLA class I molecule, and (b) a reagent fordetecting in the sample the number or cytotoxic activity of NK cells,wherein a lower protein expression level of the HLA class I molecule anda higher number or cytotoxic activity of NK cells compared to that in acontrol sample is indicative of no/low tumorigenicity of the cells orcell lines in the individual.
 14. A kit for assessing the tumorigenicityof cells or cell lines in an individual comprising: (a) a reagent fordetecting in a sample the protein expression level of an HLA class Imolecule, and (b) one or more reagents selected from the groupconsisting of: a reagent for detecting in the sample the proteinexpression level of NKp30, a reagent for detecting in the sample theprotein expression level of pERK, a reagent for detecting in the samplethe protein expression level of IL-2, and a reagent for detecting in thesample the protein expression level of IL-12, wherein a higher proteinexpression level of the HLA class I molecule and a lower proteinexpression level of NKp30, pERK, IL-2, IL-12, or any combination thereofcompared to that in a control sample is indicative of hightumorigenicity of the cells or cell lines in the individual.
 15. The kitof claim 14, wherein the expression of NKp30 indicates lowtumorigenicity of the cells or cell lines.
 16. The kit of claim 14,wherein the cells or cell lines are tumor cells of epithelial origin.